Nicotine, the principal alkaloid in tobacco, is considered to be a drug of abuse and has diverse pharmacological actions that are not completely understood. Nicotine, its metabolites, and related tobacco compounds are implicated as causative agents in diseases which are statistically more prevalent among smokers than non-smokers, e.g. lung cancer, respiratory and cardiovascular disorders, decreased fetal size and increased fetal complications. The metabolism of nicotine and related compounds is complex, and leads to the production of many metabolites. We have developed sensitive and specific assays to monitor levels of these compounds in physiological fluids, tissue extracts and in enzymatic and chemical experiments designed to determine the factors that influence the formation and catabolism of individual compounds. Specific and sensitive radioimmunoassays are available for nicotine, cotinine, Gamma(3-pyridyl)-Gamma-oxo-N-methylbutyramide, N-nitrosonornicotine, the NMN, NAD and NADP analogs of nicotine and cotinine, and N'-acylnornicotine analogs. By studying the action of these compounds in in vitro and in vivo systems, their activities can be assessed and the causes and consequences of adverse reactions better understood. Certain commonly used drugs inhibit nicotine to cotinine conversion in an in vitro system. These results suggest that special attention be given to concomitant intake of drugs when studying nicotine metabolism in man. Comparative epidemiological studies on specific populations may also reveal differences in metabolic patterns. The determination of cotinine levels in serum, urine or saliva samples is a reliable biochemical indicator of tobacco intake and is being used to validate the oral testimony given by subjects in smoking cessation programs. Emphasis is being placed on making these immunoassays more sensitive so that they can be used to obtain information about nicotine intake as a result of passive smoking. Non-radioactive immunoassays for nicotine and its metabolites will also be developed.